Plant 1-deoxy-d-xylulose 5-phosphate reductoisomerase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a 1-deoxy-D-xylulose 5-phosphate reductoisomerase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase in a transformed host cell.

This application is a continuation in part of U.S. application Ser. No.09/857,557, filed Jun. 4, 2001, and herein incorporated by reference,which is a 35 U.S.C. 371 national filing of International ApplicationNo. PCT/US99/28616, filed Dec. 3, 1999, which claims the benefit of U.S.Provisional Application No. 60/110,865, filed Dec. 4, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encoding1-deoxy-D-xylulose 5-phosphate reductoisomerase in plants and seeds.

BACKGROUND OF THE INVENTION

Isoprenoids comprise the largest family of natural products, includingnumerous secondary compounds which play different functional roles inplants as hormones, photosynthetic pigments, electron carriers, andstructural components of membranes. The fundamental unit in isoprenoidbiosynthesis, isopentenyl diphosphate (IPP), is normally synthesized bythe condensation of acetyl CoA through the mevalonate pathway. In manyorganisms including several bacteria, algae and plant plastids, IPP issynthesized by a mevalonate-independent pathway. The initial step inthis pathway is the condensation of pyruvate and glyceraldehyde3-phosphate to form 1-deoxy-D-xylulose 4-phosphate. In the committedstep towards IPP formation 1-deoxy-D-xylulose 5-phosphatereductoisomerase catalyzes in a single step an intramolecularrearrangement and reduction of 1-deoxy-D-xylulose 4-phosphate to form2-C-methyl-D-erythritol 4-phosphate.

The E. coli 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme hasonly recently been identified. Comparison of the amino acid sequence ofthe E. coli 1-deoxy-D-xylulose 5-phosphate reductoisomerase with thoseof Bacillus subtilis, Haemophilus influenzae, Helicobacter pyroli,Mycobacterium tuberculosis and Synechocystis sp. PCC6803 showed thatthere is little conservation among these sequences (Takahashi et al.(1998) Proc. Natl. Acad. Sci. USA 95:9879-9884).

SUMMARY OF THE INVENTION

The present invention concerns isolated polynucleotides comprising anucleotide sequence encoding a polypeptide having 1-deoxy-D-xylulose5-phosphate reductoisomerase activity. wherein the amino acid sequenceof the polypeptide and the amino acid sequence of SEQ ID NO:6, 8, 16,18, 20 or 24 have at least 93% sequence identity. It is preferred thatthe identity be at least 95%, it is more preferred that the identity beat least 98%. The present invention also relates to isolatedpolynucleotides comprising the complement of the nucleotide sequence.More specifically, the present invention concerns isolatedpolynucleotides encoding the polypeptide sequence of SEQ ID NO:6, 8, 16,18, 20 or 24 or nucleotide sequences comprising the nucleotide sequenceof SEQ ID NO:5, 7, 15, 17, 19 or 23.

In a first embodiment, the present invention includes an isolatedpolynucleotide comprising: (a) a nucleotide sequence encoding apolypeptide having 1-deoxy-D-xylulose 5-phosphate reductoisomeraseactivity, wherein the polypeptide has an amino acid sequence of at least93%, 95% or 98% sequence identity, based on the Clustal V method ofalignment, when compared to one of SEQ ID NO:6, 8, 16, 18, 20 or 24, or(b) a complement of the nucleotide sequence, wherein the complement andthe nucleotide sequence consist of the same number of nucleotides andare 100% complementary. The polypeptide preferably comprises the aminoacid sequence of SEQ ID NO:6, 8, 16, 18, 20 or 24. The nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:5, 7,15, 17, 19 or 23.

In a second embodiment, the present invention concerns a recombinant DNAconstruct comprising any of the isolated polynucleotides of the presentinvention operably linked to at least one regulatory sequence, and acell, a plant, and a seed comprising the recombinant DNA construct.

In a third embodiment, the present invention includes a vectorcomprising any of the isolated polynucleotides of the present invention.

In a fourth embodiment, the present invention concerns a method fortransforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention. The cell transformedby this method is also included. Advantageously, the cell is eukaryotic,e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

In a fifth embodiment, the present invention includes a method forproducing a transgenic plant comprising transforming a plant cell withany of the isolated polynucleotides of the present invention andregenerating a plant from the transformed plant cell. The invention isalso directed to the transgenic plant produced by this method, and seedobtained from this transgenic plant.

In a sixth embodiment, the present invention concerns an isolatedpolypeptide having 1-deoxy-D-xylulose 5-phosphate reductoisomeraseactivity, wherein the polypeptide has an amino acid sequence of at least93%, 95% or 98% sequence identity, based on the Clustal V method ofalignment, when compared to one of SEQ ID NO:6, 8, 16, 18, 20 or 24. Thepolypeptide preferably comprises one of SEQ ID NO:6, 8, 16, 18, 20 or24.

In a seventh embodiment, the present invention includes to a method forisolating a polypeptide having 1-deoxy-D-xylulose 5-phosphatereductoisomerase activity comprising isolating the polypeptide from acell or culture medium of the cell, wherein the cell comprises arecombinant DNA construct comprising a polynucleotide of the inventionoperably linked to at least one regulatory sequence.

In an eighth embodiment, this invention concerns a method for selectinga transformed cell comprising: (a) transforming a host cell with therecombinant DNA construct or an expression cassette of the presentinvention; and (b) growing the transformed host cell, preferably a plantcell, under conditions that allow expression of the 1-deoxy-D-xylulose5-phosphate reductoisomerase polynucleotide in an amount sufficient tocomplement a null mutant in order to provide a positive selection means.

In a ninth embodiment, this invention concerns a method of altering thelevel of expression of a 1-deoxy-D-xylulose 5-phosphate reductoisomeraseprotein in a host cell comprising: (a) transforming a host cell with arecombinant DNA construct of the present invention; and (b) growing thetransformed host cell under conditions that are suitable for expressionof the recombinant DNA construct wherein expression of the recombinantDNA construct results in production of altered levels of the1-deoxy-D-xylulose 5-phosphate reductoisomerase protein in thetransformed host cell.

A further embodiment of the instant invention is a method for evaluatingat least one compound for its ability to inhibit the activity of a1-deoxy-D-xylulose 5-phosphate reductoisomerase, the method comprisingthe steps of: (a) introducing into a host cell a recombinant DNAconstruct comprising a nucleic acid fragment encoding a1-deoxy-D-xylulose 5-phosphate reductoisomerase polypeptide, operablylinked to at least one regulatory sequence; (b) growing the host cellunder conditions that are suitable for expression of the recombinant DNAconstruct wherein expression of the recombinant DNA construct results inproduction of 1-deoxy-D-xylulose 5-phosphate reductoisomerasepolypeptide in the host cell; (c) optionally purifying the1-deoxy-D-xylulose 5-phosphate reductoisomerase polypeptide expressed byrecombinant DNA construct in the host cell; (d) treating the1-deoxy-D-xylulose 5-phosphate reductoisomerase polypeptide with a testcompound; (e) comparing the activity of the 1-deoxy-D-xylulose5-phosphate reductoisomerase polypeptide that has been treated with thetest compound to the activity of an untreated 1-deoxy-D-xylulose5-phosphate reductoisomerase polypeptide, and (f) selecting compoundswith potential for inhibitory activity based on the results of step (e)(preferably selecting the test compound if it has an inhibitory effecton the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase basedon said comparison of said step (e)).

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawing and Sequence Listing which forma part of this application.

FIGS. 1A-1C show a comparison of the amino acid sequences of the1-deoxy-D-xylulose 5-phosphate reductoisomerase polypeptides from thefollowing: a rice clone, rlr6.pk0073.d5 (SEQ ID NO:6); a corn clone,p0004.cb1hh74r (SEQ ID NO:16); a soybean contig assembled from clonesses2w.pk0029.e5, sgc3c.pk001.d16, and sr1.pk0008.d1:fis (SEQ ID NO:18);a wheat clone, wlm12.pk0003.d11:fis (SEQ ID NO:20); Arabidopsis thaliana(NCBI General Identifier No. 4886307; SEQ ID NO:21); a soybean clone,sl2.pk125.m18:fis (SEQ ID NO:24); and Mentha×piperita (NCBI GeneralIdentifier No. 77378042; SEQ ID NO:25). Amino acids conserved among allsequences are indicated with an asterisk (*) on the top row; dashes areused by the program to maximize alignment of the sequences. Amino acidresidues are numbered to the left of each sequence row and to the rightof the last sequence row. Consensus amino acid residues are numberedbelow each section of sequence alignment.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase SEQ ID NO: PlantClone Designation (Nucleotide) (Amino Acid) Corn Contig of: 1 2p0004.cb1hh74r p0012.cglac07r p0006.cbyvo28r Corn Contig of: 3 4cen3n.pk0157.e12 cr1n.pk0095.g3 cho1c.pk004.f12 csi1.pk0041.f11 Ricerlr6.pk0073.d5 5 6 Soybean Contig of: 7 8 sml1c.pk001.c15sml1c.pk005.a24 sl1.pk0021.a6 sl2.pk124.p17 sl1.pk0036.a5 sl2.pk0111.c9sl1.pk152.i19 sl2.pk0039.d4 Soybean Contig of: 9 10 sr1.pk0008.d1sr1.pk0007.c11 srm.pk0014.f8 Wheat Contig of: 11 12 wlm12.pk0003.d11wr1.pk0084.a4 Wheat Wlm24.pk0014.d7 13 14 Corn p0004.cb1hh74r 15 16Soybean Contig of: 17 18 ses2w.pk0029.e5 sgc3c.pk001.d16sr1.pk0008.d1:fis Wheat wlm12.pk0003.d11:fis 19 20 Soybeansl2.pk125.m18:fis 23 24

SEQ ID NO:21 corresponds to NCBI General Identifier No. 4886307, whichis the amino acid sequence of 1-deoxy-D-xylulose 5-phosphatereductoisomerase from Arabidopsis thaliana (Schwender et al. (1999) FEBSLett 455:140-144).

SEQ ID NO:22 corresponds to NCBI General Identifier No. 4581856, whichis the amino acid sequence of 1-deoxy-D-xylulose 5-phosphatereductoisomerase from Mentha×piperita (Lange et al. (1999) Arch BiochemBiophys 365:170-174).

SEQ ID NO:25 corresponds to NCBI General Identifier No. 77378042, whichreplaced NCBI General Identifier No. 4581856 on Oct. 7, 2005, as theamino acid sequence of 1-deoxy-D-xylulose 5-phosphate reductoisomerasefrom Mentha×piperita.

SEQ ID NO:26 corresponds to a “His-tag”, a twenty amino acid polypeptidefragment that contains six consecutive histidine residues.

SEQ ID NO:27 corresponds to a His-tagged E. coli 1-deoxy-D-xylulose5-phosphate reductoisomerase.

SEQ ID NO:28 corresponds to a His-tagged Synechocystis1-deoxy-D-xylulose 5-phosphate reductoisomerase.

SEQ ID NO:29 corresponds to a full-length His-tagged rice1-deoxy-D-xylulose 5-phosphate reductoisomerase.

SEQ ID NO:30 corresponds to a 442 amino acid truncated rice His-tagged1-deoxy-D-xylulose 5-phosphate reductoisomerase.

SEQ ID NO:31 corresponds to a 418 amino acid truncated rice His-tagged1-deoxy-D-xylulose 5-phosphate reductoisomerase.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “polynucleotide” is a nucleotide sequence such as anucleic acid fragment. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotides,of the nucleic acid sequence of the SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19, and 23.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques. A “recombinantDNA construct” comprises any of the isolated polynucleotides of thepresent invention operably linked to at least one regulatory sequence.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-a-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp (1989) CABIOS.5:151-153) and found in the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The“default parameters” are the parameters pre-set by the manufacturer ofthe program and for multiple alignments they correspond to GAPPENALTY=10 and GAP LENGTH PENALTY=10, while for pairwise alignments theyare KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Afteralignment of the sequences, using the Clustal V program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tableon the same program.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 23, and the complement ofsuch nucleotide sequences may be used in methods of selecting anisolated polynucleotide that affects the expression of a polypeptide ina plant cell. A method of selecting an isolated polynucleotide thataffects the level of expression of a polypeptide, such as1-deoxy-D-xylulose 5-phosphate reductoisomerase in a host cell(eukaryotic, such as plant or yeast, prokaryotic such as bacterial, orviral) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or theisolated chimeric gene into a host cell; measuring the level apolypeptide in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide in the host cell containing theisolated polynucleotide with the level of a polypeptide in a host cellthat does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least 70% identical,preferably at least 80% identical to the amino acid sequences reportedherein. Preferred nucleic acid fragments encode amino acid sequencesthat are at least 85% identical to the amino acid sequences reportedherein. More preferred nucleic acid fragments encode amino acidsequences that are at least 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are at least 93, 94, 95, 96, 97, 98, 99 or100% identical to the amino acid sequences reported herein. Suitablenucleic acid fragments not only have the above homologies but typicallyencode a polypeptide having at least 50 amino acids, preferably at least100 amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

The present invention includes an isolated polynucleotide comprising anucleotide sequence encoding a 1-deoxy-D-xylulose 5-phosphatereductoisomerase polypeptide having at least 93% identity, based on theClustal V method of alignment, when compared to one of SEQ ID NO:6, 8,16, 18, 20 or 24.

This invention also includes the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotide consistof the same number of nucleotides, and the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity.

Nucleic acid fragments encoding at least a portion of 1-deoxy-D-xylulose5-phosphate reductoisomerase have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other 1-deoxy-D-xylulose 5-phosphate reductoisomerases, either as cDNAs or genomic DNAs, could be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desired plantemploying methodology well known to those skilled in the art. Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan such as random primer DNAlabeling, nick translation, or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or all of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19, and 23, and the complement of such nucleotide sequences maybe used in such methods to obtain a nucleic acid fragment encoding asubstantial portion of an amino acid sequence of a polypeptide. Thepresent invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a polypeptide of a gene (suchas 1-deoxy-D-xylulose 5-phosphate reductoisomerases) preferably asubstantial portion of a plant polypeptide of a gene, comprising thesteps of: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 60 (preferably at least one of 40, mostpreferably at least one of 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, and 23, and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a portion ofa polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of plastid IPP in those cells. Because thismevalonate-independent pathway appears to be unique to microorganismsand plant plastids inhibitors of 1-deoxy-D-xylulose 5-phosphatereductoisomerases should have no affect on animals making this enzyme anexcellent herbicide candidate. Overexpression of the 1-deoxy-D-xylulose5-phosphate reductoisomerase gene will produce the active enzyme forhigh-throughput screening to find inhibitors for this enzyme. Theseinhibitors may lead to the discovery of novel herbicides.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instant polypeptideto different cellular compartments, or to facilitate its secretion fromthe cell. It is thus envisioned that the chimeric gene described abovemay be further supplemented by altering the coding sequence to encodethe instant polypeptide with appropriate intracellular targetingsequences such as transit sequences (Keegstra (1989) Cell 56:247-253),signal sequences or sequences encoding endoplasmic reticulumlocalization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.100:1627-1632) added and/or with targeting sequences that are alreadypresent removed. While the references cited give examples of each ofthese, the list is not exhaustive and more targeting signals of utilitymay be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded 1-deoxy-D-xylulose 5-phosphate reductoisomerase. An exampleof a vector for high level expression of the instant polypeptides in abacterial host is provided (Example 6).

Additionally, the instant polypeptides can be used as a targets tofacilitate design and/or identification of inhibitors of those enzymesthat may be useful as herbicides. This is desirable because thepolypeptide described herein catalyzes isopentenyl diphosphate synthesisvia the mevalonate-independent pathway. Accordingly, inhibition of theactivity of the enzyme described herein could lead to inhibition ofplant growth. Accordingly, inhibition of the activity of1-deoxy-D-xylulose 5-phosphate reductoisomerase could lead to inhibitionof plant growth. Thus, the instant polypeptides could be appropriate fornew herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean, andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat LibraryTissue Clone cen3n Corn Endosperm 20 Days After Pollination*cen3n.pk0157.e12 cho1c Corn Embryo 20 Days After Pollinationcho1c.pk004.f12 cr1n Corn Root From 7 Day Old Seedlings* cr1n.pk0095.g3csi1 Corn Silk csi1.pk0041.f11 p0004 Corn Immature Ear p0004.cb1hh74rp0006 Corn Young Shoot p0006.cbyvo28r p0012 Corn Middle 3/4 of the 3rdLeaf Blade and Mid Rib p0012.cglac07r From Green Leaves Treated withJasmonic Acid (1 mg/ml in 0.02% Tween 20) for 24 Hours Before Collectionrlr6 Rice Leaf 15 Days After Germination, 6 Hours After rlr6.pk0073.d5Infection of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistantses2w Soybean Embryogenic Suspension Two Weeks After ses2w.pk0029.e5Subculture sgc3c Soybean Cotyledon 14-21 Days After Germinationsgc3c.pk001.d16 (Starting to Turn Yellow) sl1 Soybean Two-Week-OldDeveloping Seedlings sl1.pk0021.a6 sl1 Soybean Two-Week-Old DevelopingSeedlings sl1.pk0036.a5 sl1 Soybean Two-Week-Old Developing Seedlingssl1.pk152.i19 sl2 Soybean Two-Week-Old Developing Seedlingssl2.pk0039.d4 Treated With 2.5 ppm chlorimuron sl2 Soybean Two-Week-OldDeveloping Seedlings sl2.pk0111.c9 Treated With 2.5 ppm chlorimuron sl2Soybean Two-Week-Old Developing Seedlings sl2.pk124.p17 Treated With 2.5ppm chlorimuron sl2 Soybean Two-Week-Old Developing Seedlingssl2.pk125.m18 Treated With 2.5 ppm chlorimuron sml1c Soybean Mature Leafsml1c.pk001.c15 sml1c Soybean Mature Leaf sml1c.pk005.a24 sr1 SoybeanRoot sr1.pk0008.d1 srm Soybean Root Meristem srm.pk0014.f8 wlm12 WheatSeedlings 12 Hours After Inoculation With wlm12.pk0003.d11 Erysiphegraminis f. sp tritici wlm24 Wheat Seedlings 24 Hours After InoculationWith wlm24.pk0014.d7 Erysiphe graminis f. sp tritici wr1 Wheat Root From7 Day Old Seedling wr1.pk0084.a4 *These libraries were normalizedessentially as described in U.S. Pat. No. 5,482,845, incorporated hereinby reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding 1-deoxy-D-xylulose 5-phosphate reductoisomeraseswere identified by conducting BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “p Log” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding 1-Deoxy-D-Xylulose5-Phosphate Reductoisomerase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to1-deoxy-D-xylulose 5-phosphate reductoisomerase from SynechocystisPCC6803 and Escherichia coli (NCBI General Identifier Nos. 1001556 and3434984, respectively). Shown in Table 3 are the BLAST results forindividual ESTs (“EST”), contigs assembled from two or more ESTs(“Contig”), or sequences encoding the entire protein derived from theentire cDNA inserts comprising the indicated cDNA clones (“FIS”), acontig, or an FIS and PCR (“CGS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase BLAST pLog Score CloneStatus 1001556 3434984 Contig of: Contig 14.40 10.70 p0004.cb1hh74rp0012.cglac07r p0006.cbyvo28r Contig of: Contig 111.0 59.52cen3n.pk0157.e12 cr1n.pk0095.g3 cho1c.pk004.f12 csi1.pk0041.f11rlr6.pk0073.d5 CGS 164.0 94.0 Contig of: CGS 154.0 85.50 sml1c.pk001.c15sml1c.pk005.a24 sl1.pk0021.a6 sl2.pk124.p17 sl1.pk0036.a5 sl2.pk0111.c9sl1.pk152.i19 sl2.pk0039.d4 Contig of: Contig 64.40 32.40 sr1.pk0008.d1sr1.pk0007.c11 srm.pk0014.f8 Contig of: Contig 12.70 9.30wlm12.pk0003.d11 wr1.pk0084.a4 wlm24.pk0014.d7 EST 24.70 10.70

Further sequencing of some of the above clones yielded new information.The BLASTX search using the nucleotide sequences from clones listed inTable 4 revealed similarity of the polypeptides encoded by the cDNAs to1-deoxy-D-xylulose 5-phosphate reductoisomerase from Arabidopsisthaliana, Mentha×piperita, and Synechocystis sp. (NCBI GeneralIdentifier Nos. 4886307, 4581856, and 2496789, respectively).

The 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Arabidopsisthaliana (NCBI General Identifier No. 4886307; SEQ ID NO:21), is a 406amino acid fragment that has been cloned in E. coli and shown to haveenzymatic activity (Schwender et al. (1999) FEBS Lett 455:140-144).

Shown in Table 4 are the BLAST results for the sequences of the entirecDNA inserts comprising the indicated cDNA clones (“FIS”), contigsassembled from an FIS and an EST (“Contig*”), or sequences encoding theentire protein derived from an FIS, or an FIS and PCR (“CGS”):

TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous to1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase BLAST pLog Score CloneStatus 4886307 4581856 2496789 p0004.cb1hh74rCGS >254.00 >254.00 >254.00 Contig of: CGS >254.00 >254.00 >254.00ses2w.pk0029.e5 sgc3c.pk001.d16 sr1.pk0008.d1:fis wlm12.pk0003.d11:fisFIS 145.00 145.00 145.00

Another soybean clone, sl2.pk125.m18, was identified that corresponds tothe gene of SEQ ID NOs:7 and 8. Full-insert sequencing of the soybeanclone sl2.pk125.m18 was performed and is present as SEQ ID NO:23. Thecorresponding amino acid sequence is present as SEQ ID NO:24. The FISsequence of sl2.pk125.m18 was used to identify sequencing errors in thecontig sequence of SEQ ID NO:7 (contig containing sml1c.pk001.c15), andthe corresponding amino acid sequence of SEQ ID NO:8. For SEQ ID NO:8,the amino acid residues at position 325 and 330 should have beenglutamine (Q) and isoleucine (I), respectively, and there is aframe-shift occurring after amino acid 461.

NCBI General Identifier No. 4581856 (SEQ ID NO:22), the amino acidsequence of 1-deoxy-D-xylulose 5-phosphate reductoisomerase fromMentha×piperita, was replaced on Oct. 7, 2005, by NCBI GeneralIdentifier No. 77378042 (SEQ ID NO:25).

FIGS. 1A-1C present an alignment of the amino acid sequences set forthin SEQ ID NOs:6, 16, 18, 20, and 24 and the Arabidopsis thaliana andMentha×piperita sequences (SEQ ID NO:21 and SEQ ID NO:25). The data inTable 4 represents a calculation of the percent identity of the aminoacid sequences set forth in SEQ ID NOs:6, 16, 18, 20, and 24 and theArabidopsis thaliana and Mentha×piperita sequences (SEQ ID NO:21 and SEQID NO:25).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase Percent Identity to SEQID NO: SEQ ID NO. Plant 21 (GI No. 4886307) 25 (GI No. 77378042) 6 Rice91.6 79.1 16 Corn 90.9 79.6 18 Soybean 88.4 79.6 20 Wheat 89.7 78.1 24Soybean 78.1 71.8

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode one corn, one rice, one wheat, and two soybean 1-deoxy-D-xylulose5-phosphate reductoisomerase. These sequences represent the first corn,rice, soybean, and wheat sequences encoding 1-deoxy-D-xylulose5-phosphate reductoisomerase.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (SequenaseDNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μgμL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™(Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 7 Evaluating Compounds for Their Ability to Inhibit the Activityof 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria as described in Example 6, orexpression in eukaryotic cell culture, in planta, and using viralexpression systems in suitably infected organisms or cell lines. Theinstant polypeptides may be expressed either as mature forms of theproteins as observed in vivo or as fusion proteins by covalentattachment to a variety of enzymes, proteins or affinity tags. Commonfusion protein partners include glutathione S-transferase (“GST”),thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminalhexahistidine polypeptide (“(His)₆” or “His-tag”). The fusion proteinsmay be engineered with a protease recognition site at the fusion pointso that fusion partners can be separated by protease digestion to yieldintact mature enzyme. Examples of such proteases include thrombin,enterokinase and factor Xa. However, any protease can be used whichspecifically cleaves the peptide connecting the fusion protein and theenzyme.

Purification of the instant polypeptides, if desired, may utilize anynumber of separation technologies familiar to those skilled in the artof protein purification. Examples of such methods include, but are notlimited to, homogenization, filtration, centrifugation, heatdenaturation, ammonium sulfate precipitation, desalting, pHprecipitation, ion exchange chromatography, hydrophobic interactionchromatography and affinity chromatography, wherein the affinity ligandrepresents a substrate, substrate analog or inhibitor. When the instantpolypeptides are expressed as fusion proteins, the purification protocolmay include the use of an affinity resin which is specific for thefusion protein tag attached to the expressed enzyme or an affinity resincontaining ligands which are specific for the enzyme. For example, theinstant polypeptides may be expressed as a fusion protein coupled to theC-terminus of thioredoxin. In addition, a (His)₆ peptide may beengineered into the N-terminus of the fused thioredoxin moiety to affordadditional opportunities for affinity purification. Other suitableaffinity resins could be synthesized by linking the appropriate ligandsto any suitable resin such as Sepharose-4B. In an alternate embodiment,a thioredoxin fusion protein may be eluted using dithiothreitol;however, elution may be accomplished using other reagents which interactto displace the thioredoxin from the resin. These reagents includeβ-mercaptoethanol or other reduced thiol. The eluted fusion protein maybe subjected to further purification by traditional means as statedabove, if desired. Proteolytic cleavage of the thioredoxin fusionprotein and the enzyme may be accomplished after the fusion protein ispurified or while the protein is still bound to the ThioBond™ affinityresin or other resin.

Crude, partially purified or purified enzyme, either alone or as afusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activation of theinstant polypeptides disclosed herein. Assays may be conducted underwell known experimental conditions which permit optimal enzymaticactivity. For example, assays for 1-deoxy-D-xylulose 5-phosphatereductoisomerase are presented by Kuzuyama et al. (1998) Tetrahedronlett. 39:4509-4512, Schwender et al. (1999) FEBS Lett 455:140-144, andLange et al. (1999) Arch Biochem Biophys 365:170-174.

Example 8 Expression of His-Tagged 1-Deoxy-D-Xylulose 5-PhosphateReductoisomerase Proteins in E. coli

The 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) polypeptidesfrom E. coli, Synechocystis, and rice were cloned and expressed in E.coli as histidine-tagged (His-tagged) proteins. Each His-tagged DXRpolypeptide has a twenty amino acid N-terminal extension (SEQ ID NO:26)that contains six consecutive histidine residues. E. coli expressionvectors were constructed containing polynucleotides encoding thefollowing His tagged DXR polypeptides: E. coli His-DXR (SEQ ID NO:27);Synechocystis His-DXR (SEQ ID NO:28); full-length rice His-DXR (SEQ IDNO:29); and a truncated rice His-DXR (SEQ ID NO:30), in which most ofthe putative chloroplast transit peptide (i.e., the N-terminal 51 aminoacid fragment), has been removed.

Recombinant His-tagged DXR proteins were purified from E. coli usingNi-Agarose. Recombinant DXR activity was measured using anon-radioactive kinetic assay (Kuzuyama et al. (1998) Tetrahedron Lett39:7913-7916). The assay mixture contained the following: 100 mM Hepes(pH 7.5); 1 mM MnCl₂; 0.3 nM NADPH, DXR, and 0.3 mM 1-deoxy-D-xylulose4-phosphate (DXP). The final volume of the reaction mixture was 200 μland the assay was initiated by adding the substrate. The assay was runat room temperature on a plate reader. The oxidation of NADPH wasmonitored at 340 nm. The DXR activity was determined from the slope ofthe kinetic curve.

DXR activity was detected for the E. coli His-DXR (SEQ ID NO:27) and theSynechocystis His-DXR (SEQ ID NO:28). Expression of the full-length riceHis-DXR (SEQ ID NO:29) in E. coli did not produce soluble protein in theNi-Agarose purified fraction. The full-length rice His-DXR protein (SEQID NO:29) contains the chloroplast transit peptide for the rice DXRprotein. A truncated rice His-DXR (SEQ ID NO:30), when expressed in E.coli, produced a soluble 46-kD protein in the Ni-Agarose purifiedfraction; however, no oxidation of NADPH was detected. The polypeptideof SEQ ID NO:30 consists of a rice DXR protein in which the firstfifty-one amino acids have been removed and replaced with a twenty aminoacid His-tag. A second truncated rice His-DXR polypeptide (SEQ ID NO:31)was also expressed in E. coli. SEQ ID NO:31 consists of a rice DXRprotein in which the first seventy-six amino acids have been removed andreplaced with a twenty-one amino acid His-tag. A soluble protein wasvisible in the Ni-Agarose purified fraction from E. coli expressing thesecond truncated rice His-DXR (SEQ ID NO:31); however, no oxidation ofNADPH was detected.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The disclosure of each reference set forth above is incorporated hereinby reference in its entirety.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide having 1-deoxy-D-xylulose 5-phosphatereductoisomerase activity, wherein the polypeptide has an amino acidsequence of at least 93% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO: 18, or (b) a complementof the nucleotide sequence, wherein the complement and the nucleotidesequence consist of the same number of nucleotides and are 100%complementary.
 2. The polynucleotide of claim 1, wherein the amino acidsequence of the polypeptide has at least 95% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:18.
 3. Thepolynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide has at least 98% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO:
 18. 4. Thepolynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide comprises SEQ ID NO:18.
 5. The polynucleotide of claim 1wherein the nucleotide sequence comprises SEQ ID NO:17.
 6. A vectorcomprising the polynucleotide of claim
 1. 7. A recombinant DNA constructcomprising the polynucleotide of claim 1 operably linked to at least oneregulatory sequence.
 8. A method for transforming a cell, comprisingtransforming a cell with the polynucleotide of claim
 1. 9. A cellcomprising the recombinant DNA construct of claim 7, wherein the cell isselected from the group consisting of: a yeast cell, a bacterial celland a plant cell.
 10. A method for producing a transgenic plantcomprising transforming a plant cell with the polynucleotide of claim 1and regenerating a transgenic plant from the transformed plant cell. 11.A plant comprising the recombinant DNA construct of claim
 7. 12. A seedcomprising the recombinant DNA construct of claim
 7. 13-16. (canceled)17. A method for isolating a polypeptide having 1-deoxy-D-xylulose5-phosphate reductoisomerase activity comprising isolating thepolypeptide from a cell or culture medium of the cell, wherein the cellcomprises a recombinant DNA construct comprising the polynucleotide ofclaim 1 operably linked to at least one regulatory sequence.
 18. Amethod of altering the level of expression of a 1-deoxy-D-xylulose5-phosphate reductoisomerase in a host cell comprising: (a) transforminga host cell with the recombinant DNA construct of claim 7; and (b)growing the transformed host cell under conditions that are suitable forexpression of the recombinant DNA construct wherein expression of therecombinant DNA construct results in production of altered levels of the1-deoxy-D-xylulose 5-phosphate reductoisomerase in the transformed hostcell.
 19. A method for evaluating at least one compound for its abilityto inhibit 1-deoxy-D-xylulose 5-phosphate reductoisomerase activity,comprising the steps of: (a) introducing into a host cell therecombinant DNA construct of claim 7; (b) growing the host cell underconditions that are suitable for expression of the recombinant DNAconstruct wherein expression of the recombinant DNA construct results inproduction of a 1-deoxy-D-xylulose 5-phosphate reductoisomerase; (c)optionally purifying the 1-deoxy-D-xylulose 5-phosphate reductoisomeraseexpressed by the recombinant DNA construct in the host cell; (d)treating the 1-deoxy-D-xylulose 5-phosphate reductoisomerase with a testcompound; (e) comparing the activity of the 1-deoxy-D-xylulose5-phosphate reductoisomerase that has been treated with the testcompound to the activity of an untreated 1-deoxy-D-xylulose 5-phosphatereductoisomerase, and (f) selecting the test compound if it has aninhibitory effect on the activity of 1-deoxy-D-xylulose 5-phosphatereductoisomerase based on said comparison of said step (e).